Solution phase synthesis of oligonucleotides

ABSTRACT

A process for the synthesis in solution phase of a phosphorothioate triester is provided. The process comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent to form an H-phosphonate diester. The H-phosphonate diester is oxidised in situ with a sulfur transfer agent to produce the phosphorothioate triester. Preferably, the H-phosphonate and alcohol are protected nucleosides or oligonucleotides. Oligonucleotide H-phosphonates which can be used in the formation of phosphorothioate triesters are also provided.

The present invention provides a method of synthesizing oligonucleotidesand oligonucleotide phosphorothioates in solution based on H-phosphonatecoupling and in situ sulfur transfer, carried out at Jew temperature.The invention further provides a process for the stepwise synthesis ofoligonucleotides and oligonucleotide phosphorothioates in which onenucleoside residue is added at a time, and the block synthesis ofoligonucleotides and oligonucleotide phosphorothioates in which two ormore nucleotide residues are added at a time.

In the past 15 years or so, enormous progress has been made in thedevelopment of the synthesis of oligodeoxyribonucleotides (DNAsequences), oligoribonucleotides (RNA sequences) and their analogues‘Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotidesand Analogs’, Agrawal, S. Ed., Humana Press, Totowa, 1993. Much of thework has been carried out on a micromolar or even smaller scale, andautomated solid phase synthesis involving monomeric phosphoramiditebuilding blocks Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett.,1981, 22, 1859-1862 has proved to be the most convenient approach.Indeed, high molecular weight DNA and relatively high molecular weightRNA sequences can now be prepared routinely with commercially availablesynthesisers. These synthetic oligonucleotides have met a number ofcrucial needs in biology and biotechnology.

Following Zamecnik and Stephenson's seminal discovery that a syntheticoligonucleotide could selectively inhibit gene expression in Roussarcoma virus, (Zamecnik, P.; Stephenson, M. Proc. Natl. Acad. Sci. USA1978, 75, 280-284), the idea that synthetic oligonucleotides or theiranalogues might well find application in chemotherapy has attracted agreat deal of attention both in academic and industrial laboratories.For example, the possible use of oligonucleotides and theirphosphorothioate analogues in chemotherapy has been highlighted in thereport of Gura, T. Science, 195, 270, 575-577. The so-called antisenseand antigene approaches to chemotherapy (Oligonucleotides. AntisenseInhibitors of Gene Expression, Cohen. J. S., Ed., Macmillan, Basingstoke1989 Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-649), haveprofoundly affected the requirements for synthetic oligonucleotides.Whereas milligram quantities have generally sufficed for molecularbiological purposes, gram to greater than 100 gram quantities arerequired for clinical trials. Several oligonucleotide analogues that arepotential antisense drugs are now in advanced clinical trials. If, asseems likely in the very near future, one of these sequences becomesapproved, say, for the treatment of AIDS or a form of cancer, kilogramor more probably multikilogram quantities of a specific sequence orsequences will be required.

In the past few years, a great deal of work has been carried out on thescaling-up of oligonucleotide synthesis. Virtually all of this work hasinvolved building larger and larger synthesisers and the samephosphoramidite chemistry on a solid support. The applicant is unawareof any recent improvement in the methodology of the phosphotriesterapproach to oligonucleotide synthesis in solution, which makes it moresuitable for large- and even moderate-scale synthetic work than solidphase synthesis.

The main advantages that solid phase has over solution synthesis are (i)that it is much faster, (ii) that coupling yields are generally higher,(iii) that it is easily automated and (iv) that it is completelyflexible with respect to sequence. Thus solid phase synthesis isparticularly useful if relatively small quantities of a large number ofoligonucleotides sequences are required for, say, combinatorialpurposes. However, if a particular sequence of moderate size has beenidentified and approved as a drug and kilogram quantities are required,speed and flexibility become relatively unimportant, and synthesis insolution is likely to be highly advantageous. Solution synthesis alsohas the advantage over solid phase synthesis in that block coupling(i.e. the addition of two or more nucleotide residues at a time) is morefeasible and scaling-up to any level is unlikely to present a problem.It is much easier and certainly much cheaper to increase the size of areaction vessel than it is to produce larger and larger automaticsynthesisers.

In the past, oligonucleotide synthesis in solution has been carried outmainly by the conventional phosphotriester approach that was developedin the 1970s (Reese, C. B., Tetrahedron 1978, 34, 3143-3179; Kaplan, B.E.; Itakura, K. in ‘Synthesis and Applications of DNA and RNA’, Narang,S. A., Ed., Academic Press, Orlando, 1987, pp. 9-45). This approach canalso be used in solid phase synthesis but coupling reactions aresomewhat faster and coupling yields are somewhat greater whenphosphoramidite monomers are used. This is why automated solid phasesynthesis has been based largely on the use of phosphoramidite buildingblocks; it is perhaps also why workers requiring relatively largequantities of synthetic oligonucleotides have decided to attempt thescaling-up of phosphoramidite-based solid phase synthesis.

Three main methods, namely the phosphotriester (Reese, Tetrahedron,1978), phosphoramidite (Beaucage, S. L. in Methods in Molecular Biology,Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 3-61) andH-phosphonate (Froehler, B. C. in Methods in Molecular Biology, Vol. 20,Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 63-80) approaches haveproved to be effective for the chemical synthesis of oligonucleotides.While the phosphotriester approach has been used most widely forsynthesis in solution, the phosphoramidite and H-phosphonate approacheshave been used almost exclusively in solid phase synthesis.

Two distinct synthetic strategies have been applied to thephosphotriester approach in solution.

Perhaps the most widely used strategy for the synthesis ofoligodeoxyribonucleotides in solution involves a coupling reactionbetween a protected nucleoside or oligonucleotide 3′-(2-chlorophenyl)phosphate (Chattopadhyaya, J. B.; Reese, C. B. Nucleic Acids Res., 1980,8, 2039-2054) and a protected nucleoside or oligonucleotide with a free5′-hydroxy function to give a phosphotriester. A coupling agent such as1-(mesitylene-2-sulfonyl)-3nitro-1,2,4-1H-triazole (MSNT) (Reese, C. B.;Titmas, R. C.; Yau. L. Tetrahedron Lett., 1978, 2727-2730) is required.This strategy has also been used in the synthesis of phosphorothioateanalogues. Coupling is then effected in the same way between a protectednucleoside or oligonucleotide 3′-S-(2-cyanoethyl or, for example,4-nitrobenzyl) phosphorothioate (Liu, X.; Reese, C. B. J. Chem. Soc.,Perkin Trans. 1, 1995, 1685-1695) and a protected nucleoside oroligonucleotide with a free 5′-hydroxy function. The main disadvantagesof this conventional phosphotriester approach are that some concomitant5′-sulfonation of the second component occurs (Reese, C. B.; Zhang,P.-Z. J. Chem. Soc., Perkin Trans. 1, 1995, 2291-2301) and that couplingreactions generally proceed relatively slowly. The sulfonationside-reaction both leads to lower yields and impedes the purification ofthe desired products.

The second strategy for the synthesis of oligodeoxyribonucleotides insolution involves the use of a bifunctional reagent derived from an aryl(usually 2-chlorophenyl) phosphorodichloridate and two molecularequivalents of an additive such as 1-hydroxybenzotriazole (van derMarel, et al, Tetrahedron Lett., 1981, 22, 3887-3890). A relatedbifunctional reagent, derived from 2,5-dichlorophenylphosphorodichloridothioate (Scheme 1b), has similarly been used (Kemal,O et al, J. Chem. Soc., Chem. Commun., 1983, 591-593) in the preparationof oligonucleotide phosphorothioates.

The main disadvantages of the second strategy result directly from theinvolvement of a bifunctional reagent. Thus the possibility exists ofsymmetrical coupling products being formed and the presence of smallquantities of moisture can lead to a significant diminution in couplingyields.

It is an objective of certain aspects of the present invention toprovide a new coupling procedure for the synthesis of oligonucleotidesin solution that in many embodiments (a) is extremely efficient and doesnot lead to side-reactions, (b) proceeds relatively rapidly, and (c) isequally suitable for the preparation of oligonucleotides, theirphosphorothioate analogues and chimeric oligonucleotides containing bothphosphodiester and phosphorothioate diester internucleotide linkages.

According to a first aspect of the present invention, there is provideda process for the preparation of a phosphorothioate triester whichcomprises the solution phase coupling of an H-phosphonate with analcohol in the presence of a coupling agent thereby to form anH-phosphonate diester and, in situ, reacting the H-phosphonate diesterwith a sulfur transfer agent to produce a phosphorothioate triester.

The H-phosphonate employed in the process of the present invention isadvantageously a protected nucleoside or oligonucleotide H-phosphonate,preferably comprising a 5′ or a 3′ H-phosphonate function, particularlypreferably a 3′ H-phosphonate function. Preferred nucleosides are2′-deoxyribonucleosides and ribonucleosides; preferred oligonucleotidesare oligodeoxyribonucleotides and oligoribonucleotides.

When the H-phosphonate building block is a protecteddeoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide oroligoribonucleotide derivative comprising a 3′ H-phosphonate function,the 5′ hydroxy function is advantageously protected by a suitableprotecting group. Examples of such suitable protecting groups includeacid labile protecting groups, particularly trityl and substitutedtrityl groups such as dimethoxytrityl and 9-phenylxanthen-9-yl groups;and base labile-protecting groups such as FMOC.

When the H-phosphonate building block is a protecteddeoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide oroligoribonucleotide derivative comprising a 5′ H-phosphonate function,the 3′ hydroxy function is advantageously protected by a suitableprotecting group. Suitable protecting groups include those disclosedabove for the protection of the 5′ hydroxy functions of 3′ H-phosphonatebuilding blocks and acyl, such as levulinoyl and substituted levulinoyl,groups.

When the H-phosphonate is a protected ribonucleoside or a protectedoligoribonucleotide, the 2′-hydroxy function is advantageously protectedby a suitable protecting group, for example an acid-labile acetalprotecting group, particularly1-(2-fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp); and trialkylsilylgroups, often tri(C₁₋₄-alkyl)silyl groups such as a tertiary butyldimethylsilyl group. Alternatively, the ribonuclecside oroligoribonucleotide may be a 2′-O-alkyl, 2′-O-alkoxyalkyl or2′-O-alkenyl derivative, commonly a C₁₋₄ alkyl, C₁₋₄ alkoxyC₁₋₄ alkyl oralkenyl derivative in which case, the 2′ position does not need furtherprotection.

Other H-phosphonates that may be employed in the process according tothe present invention are derived from other polyfunctional alcohols,especially alkyl alcohols, and preferably diols or triols. Examples ofalkyl diols include ethane-1,2-diol, and low molecular weightpoly(ethylene glycols), such as those having a molecular weight of up to400. Examples of alkyl triols include glycerol and butane triols.Commonly, only a single H-phosphonate function will be present, theremaining hydroxy groups being protected by suitable protecting groups,such as those disclosed hereinabove for the protection at the 5′ or 2′positions of ribonucleosides.

The alcohol employed in the process of the present invention is commonlya protected nucleoside or oligonucleotide comprising a tree hydroxygroup, preferably a free 3′ or 5′ hydroxy group, and particularlypreferably a 5′ hydroxy group.

When the alcohol is a protected nucleoside or a protectedoligonucleotide, preferred nucleosides are deoxyribonuclecsides andribonucleosides and preferred oligonucleotides areoligodeoxyribonucleotides and oligoribonucleotides.

When the alcohol is a deoxyribonucleoside, ribonucleosideoligodeoxyribonucleotide or oligoribonucleotide derivative comprising afree 5′-hydroxy group, the 3′-hydroxy function is advantageouslyprotected by a suitable protecting group. Examples of such protectinggroups include acyl groups, commonly comprising up to 16 carbon atoms,such as those derived from gamma keto acids, such as levulinoyl groupsand substituted levulinoyl groups. Substituted levulinoyl groups includeparticularly 5-halo-levulinoyl, such as 5,5,5-trifluorolevulinoyl andbenzoylpropionyl groups. Other such protecting groups include fattyalkanoyl groups, including particularly linear or branched C₆₋₁₆alkanoyl groups, such as lauroyl groups; benzoyl and substituted benzoylgroups, such as alkyl, commonly C₁₋₄ alkyl-, and halo, commonly chloroor fluoro, substituted benzoyl groups; and silyl ethers, such as alkyl,commonly C₁₋₄ alkyl, and aryl, commonly phenyl, silyl ethers,particularly tertiary butyl dimethyl silyl and tertiary butyl diphenylsilyl groups.

When the alcohol is a protected deoxyribonucleoside, ribonucleoside,oligodeoxyribonucleotides or oligoribonucleotide comprising a free3′-hydroxy group, the 5′-hydroxy function is advantageously protected bya suitable protecting group. Suitable protecting groups are thosedisclosed above for the protection of the 5′ hydroxy group ofdeoxyribonucleosides, ribonucleosides, oligodeoxyribonucleotides andoligoribonucleotide 3′ H phosphonates.

When the alcohol is a ribonuclecside or an oligoribonucleotide, the2′-hydroxy function is advantageously protected by a suitable protectinggroup, such as an acetal, particularly1-(2-fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp); and trialkylsilylgroups, often tri(C₁₋₄-alkyl)silyl groups such as a tertiary butyldimethyl silyl group. Alternatively, the ribonucleoside oroligoribonucleotide may be a 2′-O-alkyl, 2′-O-alkoxyalkyl or2-′O-alkenyl derivative, commonly a C₁₋₄ alkyl, C₁₋₄ alkoxyC₁₋₄ alkyl oralkenyl derivative, in which case, the 2′ position does not need furtherprotection.

Other alcohols that may be employed in the process according to thepresent invention are non-saccharide polyols, especially alkyl polyols,and preferably diols or triols. Examples of alkyl diols includeethane-1,2-diol, and low molecular weight poly(ethylene glycols), suchas those having a molecular weight of up to 400. Examples of alkyltriols include glycerol and butane triols. Commonly, only a single freehydroxy group will be present, the remaining hydroxy groups beingprotected by suitable protecting groups, such as those disclosedhereinabove for the protection at the 5′ or 2′ positions ofribonucleosides. However, more than one free hydroxy group may bepresent if it is desired lo perform identical couplings on more than onehydroxy group.

When the H-phosphonate and the alcohol are both protected nucleosides oroligonucleotides, the invention provides an improved method for thestepwise and block synthesis in solution of oligodeoxyribonucleotides,oligoribonucleotides and analogues thereof, based on H-phosphonatecoupling reactions. According to one preferred aspect of the presentinvention, protected nucleosides or oligonucleotides with a 3′-terminalH-phosphonate function and protected nucleosides or oligonucleotideswith a 5′-terminal hydroxy function are coupled in the presence of asuitable coupling agent to form a protected dinucleoside oroligonucleotide H-phosphonate intermediate, wherein said intermediatesundergo sulfur-transfer in situ in the presence of a suitablesulfur-transfer agent.

In addition to the presence of hydroxy protecting groups, bases presentin nucleosides/nucleotides employed in present invention are alsopreferably protected where necessary by suitable protecting groups.Protecting groups employed are those known in the art for protectingsuch bases. For example, A and/or C can be protected by benzoyl,including substituted benzoyl, for example alkyl- or alkoxy-, often C₁₋₄alkyl- or C₁₋₄ alkoxy-, benzoyl; pivaloyl; and amidine, particularlydialkylaminomethyiene, preferably di(C₁₋₄-alkyl) aminomethylene such asdimethyl or dibutyl aminomethylene. G may be protected by a phenylgroup, including substituted phenyl, for example 2,5-dichlorophenyl andalso by an isobutyryl group. T and U generally do not requireprotection, but in certain embodiments may advantageously be protected,for example at O4 by a phenyl group, including substituted phenyl, forexample 2,4-dimethylphenyl or at N3 by a pivaloyloxymethyl, benzoyl,alkyl or alkoxy substituted benzoyl, such as C₁₋₄ alkyl- or C₁₋₄alkoxybenzoyl.

When the alcohol and/or H-phosphonate is a protected nucleoside oroligonucleotide having protected hydroxy groups, one of these protectinggroups may be removed after carrying out the process of the firstinvention. Commonly, the protecting group removed is that on the3′-hydroxy function. After the protecting group has been removed, theoligonucleotide thus formed may be converted into an H-phosphonate andmay then proceed through further stepwise or block coupling and sulfurtransfers according to the process of the present invention in thesynthesis of a desired oligonucleotide sequence. The method may thenproceed with steps to remove the protecting groups from theinternucleotide linkages, the 3′ and the 5′-hydroxy groups and from thebases. Similar methodology may be applied to coupling 5′ H-phosphonates,wherein the protecting group removed is that on the 5′ hydroxy function.

In a particularly preferred embodiment, the invention provides a methodcomprising the coupling of a5′-O-(4,4′-dimethoxytrityl)-2′-deoxyribonucleoside 3′-H-phosphonate or aprotected oligodeoxyribonucleotide 3′-H-phosphonate and a component witha free 5′-hydroxy function in the presence of a suitable coupling agentand subsequent in situ sulfur transfer in the presence of a suitablesulfur-transfer agent.

In the process of the present invention, any suitable coupling agentsand sulfur-transfer agents available in the prior art may be used.

Examples of suitable coupling agents include alkyl and aryl acidchlorides, alkane and arene sulfonyl chlorides, alkyl and arylchloroformates, alkyl and aryl chlorosulfites and alkyl and arylphosphorochloridates.

Examples of suitable alkyl acid chlorides which may be employed includeC₂ to C₇ alkanoyl chlorides, particularly pivaloyl chloride. Examples ofaryl acid chlorides which may be employed include substituted andunsubstituted benzoyl chlorides, such as C₁₋₄ alkoxy, halo, particularlyfluoro, chloro and bromo, and C₁₋₄ alkyl, substituted benzoyl chlorides.When substituted, from 1 to 3 substituents are often present,particularly in the case of alkyl and halo substituents.

Examples of suitable alkanesulfonyl chlorides which may be employedinclude C₂ to C₇ alkanesulfonyl chlorides. Examples of arenesulfonylchlorides which may be employed include substituted and unsubstitutedbenzenesulfonyl chlorides, such as C₁₋₄ alkoxy, halo, particularlyfluoro, chloro and bromo, and C₁₋₄ alkyl, substituted benzenesulfonylchlorides. When substituted, from 1 to 3 substituents are often present,particularly in the case of alkyl and halo substituents.

Examples of suitable alkyl chloroformates which may be employed includeC₂ to C₁ alkyl chloroformates. Examples of aryl chloroformates which maybe employed include substituted and unsubstituted phenyl chloroformates,such as C₁₋₄ alkoxy, halo, particularly fluoro, chloro and bromo, andC₁₋₄ alkyl, substituted phenyl chloroformates. When substituted, from 1to 3 substituents are often present, particularly in the case of alkyland halo substituents.

Examples of suitable alkyl chlorosulfites which may be employed includeC₂ to C₇ alkyl chlorosulfites. Examples of aryl chlorosulfites which maybe employed include substituted and unsubstituted phenyl chlorosulfites,such as C₁₋₄ alkoxy, halo, particularly fluoro, chloro and bromo, andC₁₋₄ alkyl, substituted phenyl chlorosulfites. When substituted, from 1lo 3 substituents are often present, particularly in the case of alkyland halo substituents.

Examples of suitable alkyl phosphorochloridates which may be employedinclude di(C₁ to C₆ alkyl) phosphorochloridates. Examples of arylphosphorochloridates which may be employed include substituted andunsubstituted diphenyl phosphorochloridates, such as C₁₋₄ alkoxy, halo,particularly fluoro, chloro and bromo, and C₁₋₄ alkyl, substituteddiphenyl phosphorochloridates. When substituted, from 1 to 3substituents are often present, particularly in the case of alkyl andhalo substituents.

Further coupling agents that may be employed are the chloro-, bromo- and(benzotriazo-1-yloxy)-phosphonium and carbonium compounds disclosed byWada et al, in J.A.C.S. 1997, 119, pp 12710-12721 (incorporated hereinby reference).

Preferred coupling agents are diaryl phosphorochloridates, particularlythose having the formula (ArO)₂POCl wherein Ar is preferably phenyl,2-chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl.

The nature of the sulfur-transfer agent will depend on whether anoligonucleotide, a phosphorothioate analogue or a mixedoligonucleotide/oligonucleotide phosphorothioate is required. Sulfurtransfer agents employed in the process of the present invention oftenhave the general chemical formula:L-S-Awherein L represents a leaving group, and A represents an aryl group, amethyl or a substituted alkyl group or an alkenyl group. Commonly theleaving group is selected so as to comprise a nitrogen-sulfur bond.Examples of suitable leaving groups include morpholines such asmorpholine-3,5-dione; imides such as phthalimides, succinimides andmaleimides; indazoles, particularly indazoles with electron-withdrawingsubstituents such as 4-nitroindazoles; and triazoles.

Where a standard phosphodiester linkage is required in the finalproduct, the sulfur transfer agent, the moiety A represents an arylgroup, such as a phenyl or naphthyl group. Examples of suitable arylgroups include substituted and unsubstituted phenyl groups, particularlyhalophenyl and alkylphenyl groups, especially 4-halophenyl and4-alkylphenyl, commonly 4-(C₁₋₄ alkyl)phenyl groups, most preferably4-chlorophenyl and p-tolyl groups. An example of a suitable class ofstandard phosphodiester-directing sulfur-transfer agent is anN-(arylsulfanyl)phthalimide (succinimide or other imide may also beused).

Where a phosphorothioate diester linkage is requited in the finalproduct, the moiety A represents a methyl, substituted alkyl or alkenylgroup. Examples of suitable substituted alkyl groups include substitutedmethyl groups, particularly benzyl and substituted benzyl groups, suchas alkyl-, commonly C₁₋₄alkyl- and halo-, commonly chloro-, substitutedbenzyl groups, and substituted ethyl groups, especially ethyl groupssubstituted at the 2-position with an electron-withdrawing substituentsuch as 2-(4-nitrophenyl)ethyl and 2-cyanoethyl groups. Examples ofsuitable alkenyl groups are allyl and crotyl. Examples of a suitableclass of phosphorothioate-directing sulfur-transfer agents are, forexample, (2-cyanoethyl)sulfanyl derivatives such as4-[(2-cyanoethyl)-sulfanyl]morpholine-3,5-dione or a correspondingreagent such as 3-(phthalimidosulfanyl)propanonitrile.

A suitable temperature for carrying out the coupling reaction and sulfurtransfer is in the range of approximately −55° C. to room temperature(commonly in the range of from 10 to 30° C., for example approximately20° C.), and preferably from −40° C. to 0° C.

Organic solvents which can be employed in the process of the presentinvention include haloalkanes, particularly dichloromethane, esters,particularly alkyl esters such as ethyl acetate, and methyl or ethylpropionate, and basic, nucleophilic solvents such as pyridine. Preferredsolvents for the coupling and sulfur transfer steps are pyridine,dichloromethane and mixtures thereof.

The mole ratio of H-phosphonate to alcohol in the process of the presentinvention is often selected to be in the range of from about 0.9:1 to3:1, commonly from about 1:1 to about 2:1, and preferably from about1.1:1 to about 1.5:1, such as about 1.2:1. However, where couplings onmore than one free hydroxyl are taking place at the same time, the moleratios will be increased proportionately. The mole ratio of couplingagent to alcohol is often selected to be in the range of from about 1:1to about 10:1, commonly from about 1.5:1 to about 5:1 and preferablyfrom about 2:1 to about 3:1. The mole ratio of sulfur transfer agent toalcohol is often selected to be in the range of from about 1:1 to about10:1, commonly from about 1.5:1 to about 5:1 and preferably from about2:1 to about 3:1.

In the process of the present invention, the H-phosphonate and thealcohol can be pre-mixed in solution, and the coupling agent added tothis mixture. Alternatively, the H-phosphonate and the coupling agentcan be pre-mixed, often in solution and then added to a solution of thealcohol, or the alcohol and the coupling agent may be mixed, commonly insolution, and then added to a solution of the H-phosphonate. In certainembodiments, the H-phosphonate, optionally in the form of a solution,can be added to a solution comprising a mixture of the alcohol and thecoupling agent. After the coupling reaction is substantially complete,the sulfur transfer agent is then added to the solution theH-phosphonate diester produced in the coupling reaction. Reagentadditions commonly take place continuously or incrementally over anaddition period.

In the process of the present invention, it is possible to prepareoligonucleotides containing both phosphodiester and phosphorothioatediester internucleotide linkages in the same molecule by selection ofappropriate sulfur transfer agents, particularly when the process iscarried out in a stepwise manner.

As stated previously, the method of the invention can be used in thesynthesis of RNA, 2′-O-alkyl-RNA, 2′-O-alkoxyalkyl-RNA and2′-O-alkenyl-RNA sequences. 2′-O-(F pmp)-5′-O-(4,4-dimethoxytrityl)-ribonucleoside 3′-H-phosphonates 1 and 2′-O-(alkyl,alkoxyalkyl or alkenyl)-5′-O-(4,4-dimethoxytrityl)-ribonuclecside3′-H-phosphonates 2a-c may be prepared, for example, from thecorresponding nucleoside building blocks, ammonium p-cresylH-phosphonate and pivaloyl chloride.

The same protocols are used as in the synthesis of DNA and DNAphosphorothioate sequences (Schemes 2-4). Following the standardunblocking procedure (Scheme 2, steps v and vi), the Fpmp protectinggroups are removed under mild conditions of acidic hydrolysis that leadto no detectable cleavage or migration of the internucleotide linkages(Capaldi, D. C.; Reese, C. B. Nucleic Acids Res. 1994, 22, 2209-2216).For chemotherapeutically useful ribozyme sequences, relatively largescale RNA synthesis in solution is a matter of considerable practicalimportance. The incorporation of 2′-O-alkyl, 2′-O-substituted alkyl and2′-O-alkenyl [especially 2′-O-methyl, 2′-O-allyl and2′-O-(2-methoxyethyl)]-ribonucleosides (Sprcat, B. S. in ‘Methods inMolecular Biology, Vol. 20. Protocols for Oligonucleotides and Analogs’,Agrawal, S., Ed., Humana Press, Totowa, 1993) into oligonucleotides iscurrently a matter of much importance as these modifications confer bothresistance to nuclease digestion and good hybridisation properties onthe resulting oligomers.

The sulfur transfer step is carried out on the product of theH-phosphonate coupling in situ, ie without separation and purificationof the intermediate produced by the coupling reaction. Preferably, thesulfur transfer agent is added to the stirred mixture resulting from thecoupling reaction.

In addition to the fact that it is carried out in homogenous solution,the present coupling procedure differs from that followed in theH-phosphonate approach to solid phase synthesis (Froehler et al.,Methods in Molecular Biology, 1993) in at least two other importantrespects. First, it may be carried out at a very low temperature. Sidereactions which can accompany H-phosphonate coupling (Kuyl-Yeheskiely etal, Rec. Trav. Chim., 1986, 105, 505-506) can thereby be avoided evenwhen di-(2-chlorophenyl) phosphorodichloridate rather than pivaloylchloride (Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett., 1986, 27,469-472) is used as the coupling reagent. Secondly, sulfur transfer iscarried out after each coupling step rather than just once following theassembly of the whole oligomer sequence.

Protecting groups can be removed using methods known in the art for theparticular protecting group and function. For example, transientprotecting groups, particularly gamma keto acids such as levulinoyl-typeprotecting groups, can be removed by treatment with hydrazine, forexample, buffered hydrazine, such as the treatment with hydrazine undervery mild conditions disclosed by van Boom. J. H.; Burgers, P. M. J.Tetrahedron Lett., 1976, 4875-4878. The resulting partially-protectedoligonucleotides with free 3′-hydroxy functions may then be convertedinto the corresponding H-phosphonates which are intermediates which canbe employed for the block synthesis of oligonucleotides and theirphosphorothioate analogues.

When deprotecting the desired product once this has been produced,protecting groups on the phosphorus which produce phosphorothioatetriester linkages are commonly removed first. For example, a cyanoethylgroup can be removed by treatment with a strongly basic amine such asDABCO, 1,5-diazabicylo[4.3.0]non-5-ene (DBN),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine.

Phenyl and substituted phenyl groups on the phosphorothioateinternucleotide linkages and on the base residues can be removed byoximate treatment, for example with the conjugate base of an aldoxime,preferably that of E-2-nitrobenzaldoxime or pyridine-2-carboxaldoxime(Reese et al. Nucleic Acids Res. 1981). Kamimura, T. et al in J. Am.Chem. Soc., 1984, 106 4552-4557 and Sekine. M. Et al, Tetrahedron, 1985,41, 5279-5288 in an approach to oligonucleotide synthesis by thephosphotriester approach in solution, based on S-phenyl phosphorothioateintermediates: and van Boom and his co-workers in an approach tooligonucleotide synthesis, based on S-(4-methylphenyl) phosphorothioateintermediates (Wreesman, C. T. J. Et al, Tetrahedron Lett., 1985, 26,933-936) have all demonstrated that unblocking S-phenylphosphorothioateswith oximate ions (using the method of Reese et al., 1978; Reese, C. B,;Zard, L. Nucleic Acids Res., 1981, 9, 4611-4626) led to naturalphosphodiester internucleotide linkages. In the present invention, theunblocking of S-(4-chlorophenyl)-protected phosphorothioates with theconjugate base of E-2-nitrobenzaldoxime proceeds smoothly and with nodetectable internucleotide cleavage.

Other base protecting groups, for example benzoyl, pivaloyl and amidinegroups can be removed by treatment with concentrated aqueous ammonia.

Trityl groups present can be removed by treatment with acid. With regardto the overall unblocking strategy in oligodeoxyribonucleotidesynthesis, another important consideration of the present invention, isthat the removal of trityl, often a 5′-terminal DMTr, protecting group(‘detritylation’) should proceed without concomitant depurination,especially of any 6-N-acyl-2′-deoxyadenosine residues. According to anembodiment of the invention, the present inventors have found that suchdepurination, which perhaps is difficult completely to avoid in solidphase synthesis, can be totally suppressed by effecting ‘detritylation’with a dilute solution of hydrogen chloride at low temperature,particularly ca. 0.45 M hydrogen chloride in dioxane-dichloromethane(1:8 v/v) solution at −50° C. Under these reaction conditions,‘detritylation’ can be completed rapidly, and in certain cases after 5minutes or less. For example, when6-N-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine was treatedwith hydrogen chloride in dioxane-dichloromethane under such conditions,‘detritylation’ was complete after 2 min, but no depurination wasdetected even after 4 hours.

Silyl protecting groups may be removed by fluoride treatment, forexample with a solution of a tetraalkyl ammonium fluoride salt such astetrabutyl ammonium fluoride.

Fpmp protecting groups may be removed by acidic hydrolysis under mildconditions.

This new approach to the synthesis of oligonucleotides in solution issuitable for the preparation of sequences with (a) solelyphosphodiester, (b) solely phosphorothioate diester and (c) acombination of both phosphodiester and phosphorothioate diesterinternucleotide linkages.

The invention also relates to the development of block coupling (asillustrated for example in Scheme 4b). In this respect, the examplesprovide an illustration of the synthesis ofd[Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T] (ISIS 5320 Ravikuma, V. T.,Cherovallath, Z. S. Nucleosides & Nucleosides 1996, 15, 1149-1155), anoctadeoxyribonucleoside heptaphosphorothioate, from tetramer blocks.This oligonucleotide analogue has properties as an anti-HIV agent. Otherproposed block synthesis targets include sequences with therapeuticeffects, for example, inhibitors of human thrombin and anti-HIV agents.The method of the invention furthermore can be used in the synthesis oflarger sequences.

It will be apparent that when the process of the present invention isapplied to block synthesis, a number of alternative strategies areavailable in terms of the route to the desired product. These willdepend on the nature of the desired product. For example, an octamer maybe prepared by the preparation of dimers, coupled to produce tetramers,which are then coupled to produce the desired octamer. Alternatively, adimer and a trimer may be coupled to produce a pentamer, which can becoupled with a further trimer to produce the desired octamer. The choiceof strategy is at the discretion of the user. However, the commonfeature of such block coupling is that an oligomer H-phosphonatecomprising two or more units is coupled with an oligomer alcohol alsocomprising two or more units. Most commonly oligonucleotide3′-H-phosphonates are coupled with oligonucleotides having free5′-hydroxy functions.

The process of the present invention can also be employed to preparecyclic oligonucleotides, especially cyclic olicodeoxyribonucleotides andcyclic ribonucleotides. In the preparation of cyclic oligonucleotides,an oligonucleotides comprising an H-phosphonate function, often a 3′ or5′ H-phosphonate is prepared, and a free hydroxy function is introducedby appropriate deprotection. The position of the free hydroxy functionis usually selected to correspond to the H-phosphonate, for example a 5′hydroxy function would be coupled with a 3′ H-phosphonate, and a 3′hydroxy function would be coupled with a 5′ H-phosphonate. The hydroxyand the H-phosphonate functions can then be coupled intramolecularly insolution in the presence of a coupling agent and this reaction isfollowed by in situ sulfur transfer.

According to a further aspect of the present invention, there isprovided novel oligomer H-phosphonates having the general chemicalformula:

wherein

-   -   each B independently is a base selected from A, G, T C or U;    -   each Q independently is H or OR′ wherein R′ is alkyl,        substituted alkyl, alkenyl or a protecting group;    -   each R independently is an aryl, methyl, substituted alkyl or        alkenyl group;    -   W is H, a protecting group or an H-phosphonate group of formula        in which M⁺ is a monovalent cation;    -   each X independently represent O or S;    -   each Y independently represents O or S;    -   Z is H, a protecting group or an H-phosphonate group of formula        in which M⁺ is a monovalent cation; and    -   n is a positive integer;    -   provided that when W is H or a protecting group, that Z is an        H-phosphonate group, and    -   that when Z is H or a protecting group, that W is an        H-phosphonate group.

Preferably, only one of W or Z is an H-phosphonate group, commonly onlyZ being an H-phosphonate group.

When W or Z represents a protecting group, the protecting group may beone of those disclosed above for protecting the 3′ or 5′ positionsrespectively. When W is a protecting group, the protecting group is atrityl group, particularly a dimethoxytrityl group. When Z is aprotecting group, the protecting group is a trityl group, particularly adimethoxytrityl group, or an acyl, preferably a levulinoyl group.

The bases A, G and C represented by B are preferably protected, andbases T and U may be protected. Suitable protecting groups include thosedescribed hereinabove for the protection of bases in the processaccording to the first aspect of the present invention.

When Q represents a group of OR′, and R′ is alkenyl, the alkenyl groupis often a C₁₋₄ alkenyl group, especially allyl or crotyl group. When R′represents alkyl, the alkyl is preferably a C₁₋₄ alkyl group. When R′represents substituted alkyl, the substituted alkyl group includesalkoxyalkyl groups, especially C₁₋₄ alkyoxyC₁₋₄ alkyl groups such asmethoxyethyl groups. When R′ represents a protecting group, theprotecting group is commonly an acid-labile acetal protecting group,particularly 1-(2-fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp) or atrialkylsilyl groups, often a tri(C₁₋₄-alkyl)silyl group such as atertiary butyl dimethylsilyl group.

Preferably, X represents O.

In many embodiments, Y represents S and each R represents the methyl,substituted alkyl, alkenyl or aryl group remaining from the sulfurtransfer agent(s) employed in the process of the present invention.Preferably, each R independently represents a methyl group; asubstituted methyl group, particularly a benzyl or substituted benzylgroup, such as an alkyl-, commonly C₁₋₄alkyl- or halo-, commonlychloro-, substituted benzyl group; a substituted ethyl group, especiallyan ethyl group substituted at the 2-position with anelectron-withdrawing substituent such as a 2-(4-nitrophenyl)ethyl or a2-cyanoethyl group: a C₁₋₄ alkenyl croup, preferably an allyl and crotylgroup; or a substituted or unsubstituted phenyl group, particularly ahalophenyl or alkylphenyl group, especially 4-halophenyl group or a4-alkylphenyl, commonly a 4-(C₁₋₄ alkyl)phenyl group, and mostpreferably a 4-chlorophenyl or a p-tolyl group.

M+ preferably represents a trialkyl ammonium ion, such as atri(C₁₋₄-alkylammonium) ion, and preferably a triethylammonium ion.

n may be 1 up to any number depending on the oligonucleotide which isintended to be synthesised, particularly up to about 20. Preferably n is1 to 16, and especially 1 to 9. H-phosphonate wherein n represents 1, 2or 3 can be employed when it is desired to add small blocks ofnucleotide, with correspondingly larger values of n, for example 5, 6 or7 or mote being employed if larger blocks of oligonucleotide are desiredto be coupled.

The H-phosphonates according to the present invention are commonly inthe form of solutions, preferably these employed in the process of thefirst aspect of the present invention.

These H-phosphonates are also useful intermediates in the blocksynthesis of oligonucleotides arid oligonucleotide phosphorothioates. Asindicated above, block coupling is much more feasible in solution phasethan in solid phase synthesis.

The oligonucleotide H-phosphonates can be prepared using general methodsknown in the art for the synthesis of nucleoside H-phosphonates.Accordingly, in a further aspect of the present invention, there isprovided a process for the production of an oligonucleotideH-phosphonate wherein an oligonucleotide comprising a free hydroxyfunction, preferably a 3′ or 5′ hydroxy function, is reacted with analkyl or aryl H-phosphonate salt in the presence of an activator.

Preferably, the oligonucleotide is a protected oligonucleotide, and mostpreferably a protected oligodecoxynucleotide or a protectedoligoribonucleotide. The H-phosphonate salt is often an ammonium salt,including alkyl, aryl and mixed alkyl and aryl ammonium salts.Preferably, the ammonium salt is an (NH₄)⁺ or a tri(C₁₋₄alkyl) ammoniumsalt. Examples of alkyl groups which may be present in the H-phosphonateare C₁₋₄ alkyl, especially C₂₋₄ alkyl, groups substituted with stronglyelectron withdrawing groups, particularly halo, and preferably fluorogroups, such as 2,2,2-trifluoroethyl and1,1,1,3,3,3-hexafluoropropan-2-yl groups. Examples of aryl groups whichmay be present include phenyl and substituted phenyl, particularlyalkylphenyl, commonly C₁₋₄ alkylphenyl and halophenyl, commonlychlorophenyl groups. Preferably, a substituted phenyl group is a4-substituted phenyl group. Particularly preferred H-phosphonates areammonium and triethylammonium p-cresyl H-phosphonates. Activators whichmay be employed include those compounds disclosed herein for use ascoupling agents, and particularly diaryl phosphorochloridates and alkyland cycloalkyl acid chlorides, such as 1-adamantanecarbonyl chloride,and preferably pivaloyl chloride. The production of H-phosphonatespreferably takes place in the presence of a solvent often those solventsdisclosed for use in the process of the first aspect of the presentinvention, preferably pyridine, dichloromethane and mixtures thereof.

One advantage of the present invention for the synthesis of solelyphosphorothioate diesters is that, provided care is taken to avoiddesulfurisation during the unblocking steps [particularly during heatingwith aqueous ammonia (for example Scheme 3. step viii(a))], thesynthesis of oligonucleotide phosphorothioates should not lead toproducts that are contaminated with standard phosphodiesterinternucleotide linkages. In the case of solid phase oligonucleotidephosphorothioate synthesis, incomplete sulfur transfer in each syntheticcycle usually leads to a residual phosphodiester contamination (Zon, G.;Stec, W. J. in ‘Oligonucleotides and Analogs. A Practical Approach’,Eckstein, F., Ed., IRL Press. Oxford, 1991, pp. 87-108).

The solution synthesis as proposed by the present invention has anotherenormous advantage over solid-phase synthesis in that the possibilityexists of controlling the selectivity of reactions by working at low oreven at very low temperatures. This advantage extends to thedetritylation step (Scheme 3, step i) which can proceed rapidly andquantitatively below 0° C. without detectable depurination. After thedetritylation step, a relatively quick and efficient purification can beeffected by what has previously been described as the ‘filtration’approach (Chaudhuri, B,; Reese, C. B.; Weclawek, K. Tetrahedron Lett.1984, 25, 4037-4040). This depends on the fact that phosphotriester (andphosphorothioate triester) intermediates, but not any remainingdetritylated charged monomers, are very rapidly eluted from shortcolumns of silica gel by THF-pyridine mixtures.

The method according to the invention will now be illustrated withreference to the following examples which are not intended to belimiting:

In the Examples, it should be noted, that where nucleoside residues andinternucleotide linkages are italicised, this indicates that they areprotected in some way. In the present context, A, C, G, and T represent2′-deoxyadenosine protected on N-6 with a benzoyl group,2′-deoxycytidine protected on N-4 with a benzoyl group,2′-deoxyguanosine protected on N-2 and on O-6 with isobutyryl and2,5-dichlorophenyl groups and unprotected thymine. For example, asindicated in scheme 3, p(s) and p(s′) represent S-(2-cyanoethyl) andS-(4-chlorophenyl) phosphorothioates, respectively, and p(H), which isnot protected and therefore not italicised, represents an H-phosphonatemonoester if it is placed at the end of a sequence or attached to amonomer but otherwise it represents an H-phosphonate diester.

EXAMPLES Reaction Scheme for Preparation of Dinucleoside Phosphates

With particular reference to the preparation of dinucleoside phosphates,Scheme 2 describes in more detail the method of the invention for thepreparation of olicodeoxyribonucleotides and the phosphorothioateanalogues thereof.

Reagents and Conditions

(i) 18, C₅H₅N, CH₂Cl₂, −40° C., 5-10 min;

(ii) 19, C₅H₅N, CH₂Cl₂, −40° C., 15 min, b, C₅H₅N—H₂O (1:1 v/v), −40° C.to room temp.

(iii) 4 M HCl/dioxane, CH₂Cl₂, −50° C., 5 min;

(iv) Ac₂O, C₅H₅N, room temp., 15 h;

(v) 20, TMG, MeCN, room temp., 12 h;

(vi) a, conc. aq. NH₃ (d 0.88), 50° C., 15 h, b, Amberlite IR-120(plus), Na+ form, H₂O;

(vii) a, 21, C₅H₅N, CH₂Cl₂, −40° C., 15 min, b, C₅H₅N—H₂O (1:1 v/v),−40° C. to room temp.;

(viii) DBU, Me₃SiCl, CH₂Cl₂, room temp., 30 min;

(ix) 20, DBU, MeCN, room temp., 12 h.

From Scheme 2, the synthesis of oligonucleotides proceeds throughintermediates 8, 9, 10 and 11 and the preparation of thephosphorothioate analogues proceeds through intermediates 8, 9, 12 and13. Eases 14, 16 and 16 correspond to protected adenine, protectedcytosine and protected guanine. Base 17 corresponds to thymine whichdoes not require protection. Any conventionally used protecting groupcan be used. In the synthesis of RNA, thymine will be replaced byuracil. Compound 18 is a suitable coupling agent, and compounds 19 and21 are suitable sulfur transfer agents. These compounds are referred tomore fully hereinbelow.

The monomeric building blocks required in the coupling procedureaccording to the invention illustrated in Scheme 2 are triethylammonium5′-O-(4-4′-dimethoxytrityl)-2′-deoxyribonucleoside 3′-H-phosphonates 8(Eases B and B′=14-17) which can readily be prepared in almostquantitative yields from the corresponding protected nucleosidederivatives by a recently reported procedure (Ozola. V., Reese. C. B.,Song Q. Tetrahedron Lett., 1996, 37, 8621-8624). By way of illustration,triethylammonium 5′-O-(dimethoxytrityl)-2′-deoxyribonucleoside3′-H-phosphonates 8 were prepared as follows: Ammonium 4-methylphenylH-phosphonate 30 (2.84 g, 15.0 mmol),5′-O-(dimethyoxytrityl)-2′-deoxyribonucleoside derivative (5.0 mmol),triethylamine (4.2 ml, 30 mmol) and dry pyridine (20 ml) were evaporatedtogether under reduced pressure. The residue was coevaporated again withdry pyridine (20 ml). The residue was dissolved in dry pyridine (40 ml)and the solution was cooled to −35° C. (industrial methylatedspirits/dry ice bath). Pivaloyl chloride (1.85 ml, 15.0 mmol) was addeddropwise to the stirred solution over a period of 1 min, and thereactants were maintained at −35° C. After 30 min, water (5 ml) wasadded, and the stirred mixture was allowed to warm up to roomtemperature. Potassium phosphate buffer (1.0 mol dm⁻³, pH 7.0, 250 ml)was added to the products, and the resulting mixture was concentratedunder reduced pressure until all of the pyridine had been removed. Theresidual mixture was partitioned between dichloromethane (250 ml) andwater (200 ml). The organic layer was washed with triethylammoniumphosphate buffer (0.5 mol dm⁻³, pH 7.0, 3×50 ml), dried (MgSO₄) and thenevaporated under reduced pressure. The reside was fractionated by shortcolumn chromatography on silica gel (25 g). Appropriate fractions,eluted with dichloromethane-methanol (95:5 to 90:10 v/v), wereevaporated to give (5′-O-(dimethoxytrityl)-2′-deoxyribonucleoside3′-H-phosphonate 8.

When triethylammonium6-N-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deooxyadenosine3′-H-phosphonate (DMTr-Ap(H))(Ozo/a et al., Tetrahedron, 1996), 8(B-14), 4-N-benzoyl-3′-O-levulinoyl-2′-deoxycytidine (HO-C-Lev) 9(B′=15) and di-(2-chlorophenyl) phosphorochloridate 18 were allowed toreact together in pyridine-dichloromethane solution at −40° C., thecorresponding fully-protected dinucleoside H-phosphonate(DMTr-Ap(H)C-Lev) was obtained apparently in quantitative yield within5-10 minutes. The protocol used in this particular example was thedropwise addition of a solution of di-(2-chlorophenyl)phosphorochloridate (2.03 g, 6.0 mmol) in dichloromethane (4 ml) over 5min to a stirred, dry solution of the triethylammonium salt ofDMTr-Ap(H) 8 (B=14) (3.95 g, ca. 4.8 mmol) and4-N-benzoyl-3′-O-levulinoyl-2′-deoxycytidine 9 (B′=15) (1.72 g, 4.0mmol) in pyridine (36 ml), maintained at −40° C. (industrial methylatedspirits+dry ice bath). After a further period of 5 min. only onenucleotide product assumed to be DMTr-Ap(H)C-Lev. and some remainingH-phosphonate monomer 8 (B=14) could be detected by reverse phase HPLC).However, it should be noted that these reaction conditions can be variedappropriately.

It is particularly noteworthy that such a high coupling efficiency wasachieved with only ca. 20% excess of H-phosphonate monomer. No attemptwas made to isolate the intermediate dinucleoside H-phosphonate(DMTr-Ap(H)C-Lev).

N-[(4-Chlorophenyl)sulfanyl]phthalimide 19 (2.32 g, 8.0 mmol)(Behforouz, M.; Kerwood, J. E. J. Org. Chem, 1969, 34, 51-55) was addedto the stirred reactants which were maintained at −40° C. After 15 min.the products were worked up and chromatographed on silica gel and thecorresponding S-(4-chlorophenyl) dinucleoside phosphorothioateDMr-Ap(s)C-Lev 10 (E=14, B′=15) was obtained in ca. 99% isolated yield.Thus both coupling and the sulfur-transfer steps proceeded relativelyquickly and virtually quantitatively at −40° C.

The four step procedure (Scheme 2, steps iii-vi) for the unblocking ofDMTr-Ap(s′)C-Lev 10 (E=14. B′=15) preferably involves ‘detritylation’,acetylation of the 5′-terminal hydroxy function, oximate treatment, andfinally treatment with concentrated aqueous ammonia to remove acylprotecting groups from the base residues and from the 3′- and5′-terminal hydroxy functions. In this way, extremely pure d[ApC] 11(B=adenin-9-yl, B′=cytosine-1-yl) was obtained without furtherpurification and isolated as its sodium salt. The monomeric buildingblocks 8 (B=17) and 9 (B′=16) were coupled together in the same way andon the same scale. After sulfur transfer withN-[(4-chlorophenyl)sulfanyl]phthalimide 19, the fully protecteddinucleoside phosphorothioate DMTr-Tp(s)G-Lev 10 (B=17, B′16) wasisolated in ca. 98% yield. Again, very pure d[TpG] 11 (B=thymin-1-yl,B′=guanin-9-yl) was obtained when this material was unblocked by theabove procedure (Scheme 2, steps iii-vi).

The protocol for the preparation of fully-protected oligonucleotidephosphorothioates differs from that used for oligonucleotide synthesisonly in that sulfur-transfer is effected with4-[(2-cyanoethyl)sulfanyl]morpholine-3,5-dione 21 or3-(phthalimidosulfanyl)propanonitrile. However,4-[(2-cyanoethyl)sulfanylgmorpholine-3,5-dione has the advantage thatthe morpholine-3,5-dione produced in the course of sulfur-transfer ismore water-soluble than phthalimide. Triethylammonium6-O-(2,5-dichlorophenyl)-5′-O-(4,4′-dimethoxytrityl)-2-N-isobutyryl-2′-deoxyguanosine3′-H-phosphonate (DMTr-Gp(H)) 8 (B=16) [ca. 4.8 mmol],6-N-benzoyl-3′-O-levulinoyl-2′-deoxyadenosine (HO-A-Lev) 9 (B=14) 14.0mmol) and di-(2-chlorophenyl) phosphorochloridate 18 [6.0 mmol] wereallowed to react together in pyridine-dichloromethane solution at −40°C. for 5-10 minutes. 4-[(2-Cyanoethyl)sulfanyl]morpholine-3,5-dione 21[8.0 mmol] (Scheme 2, step vii) was then added while the reactants weremaintained at −40° C. After 15 minutes, the products were worked up andfractionated by chromatography on silica gel to give the fully-protecteddinucleoside phosphorothioate (DMTr-Gp(s)A-Lev) 12 (B=14. B′=16) in 99%isolated yield. This material was unblocked by a five-step procedure(Scheme 2, steps iii, iv, viii, ix and vi). Following the‘detritylation’ and acetylation steps, the product was treated with1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) under strictly anhydrousconditions to remove the S-(2-cyanoethyl) protecting group. The6-O-(2,5-dichlorophenyl) protecting group was then removed from theguanine residue by oximate treatment, and finally all of the acylprotecting groups were removed by ammonolysis. The oximate treatmentstep can be omitted if the oligonucleotide phosphorothioate does notcontain any 2′-deoxyguanosine residues. Extremely pure d[Gp(s)A] 13(B=guanin-9-yl. B′=adenin-9-yl) was obtained without furtherpurification, and was isolated as its sodium salt.

Preparation of 4-[(2-cyanoethyl)sulfanyl]morpholine-3,5-dione

S-(2-cyanoethyl)isothiouronium chloride was prepared as follows.Thiourea (304 g), was dissolved with heating in concentratedhydrochloric acid (500 ml). The resulting solution was evaporated underreduced pressure and the residual colourless solid was dissolved inboiling absolute ethanol (1300 ml). The solution was cooled to roomtemperature and acrylonitrile (400 cm³) was added in portions withstirring. The reactants were heated, under reflux, for 2 hours. Thecooled products were filtered and the residue was washed with coldethanol and then dried in vacuo over calcium chloride.

Di-(2-cyanoethyl) disulphide was then prepared as follows.Dichloromethane (400 ml) was added to a stirred solution ofS-(2-cyanoethyl)isothiouronium chloride (83.0 g) in water (500 ml) at 0°C. (ice-water bath). Sodium perborate tetrahydrate (44.1 g) was added,and then a solution of sodium hydroxide (30.0 g) in water (250 ml) wasadded dropwise. The reactants were maintained at 0° C. (ice-water bath).After 5 hours, the products were separated and the aqueous layer wasextracted with dichloromethane (3×50 ml). The combined organic layerswere dried (MgSO₄) and evaporated under reduced pressure to give a solidwhich was recrystallised from methanol (30 ml) to give colourlesscrystals.

Di-(2-cyanoethyl)disulphide (4.51 g) and morpholin-2,6-dione (5.75 g)were suspended in acetonitrile (10 ml), dichloromethane (20 ml) and2.6-lutidine (17.4 ml) and cooled to 0° C. (ice-water Lath). A solutionof bromine (4.28 g) in dichloromethane (20 ml) was added over 30minutes. The reaction mixture was allowed to stir at 0° C. For 1.5hours. The product was then precipitated by the addition of ice-coldmethanol (50 ml) over 30 minutes and filtered to give the title compound(8.28 g, 82%). Recrystallisation from ethyl acetate cave4-[(2-cyanoethyl)sulfanyl]morpholine-3,5-dione as colourless needles,m.p. 121-122° C.

Reaction Scheme for Preparation of Chimeric Oligonucleotides

The stepwise synthesis of d[pGp(s)ApC] 25 which has one phosphorothioatediester and two phosphodiester internucleotide linkages is illustratedin outline by way of example in Scheme 3.

Reagents and Conditions

(i) 4 M HCl/dioxane, CH₂Cl₂, −50° C., 5 min;

(ii) 18, C₅H₅N, CH₂Cl₂, −40° C., 5-10 min;

(iii) a, 21, C₅H₅N, CH₂Cl₂, −40° C., 15 min, b, C₅H₅N—H₂O (1:1 v/v),−40° C. to room temp;

(iv) a, 19, C₅H₅N, CH₂Cl₂, 40° C., 15 min, b, C₅H₅N—H₂O (1:1 v/v), −40°C. to room temp;

(v) Ac₂O, C₅H₅N, room temp., 15 h;

(vi) DBU, Me₂SiCl, CH₂Cl₂, room temp., 30 min:

(vii) 20, DBU, MeCN, room temp., 12 h;

(viii) a, conc. aq. NH₃(d 0.88), 50° C., 15 h, b, Ambcriltle IR-120(plus), Na+ form. H₂O.

No limitation of scale is anticipated. The reactions shown in Scheme 3are not intenced to be limiting and the method of the invention isequally suitable for the synthesis of RNA, 2′-O-alkyl-RNA and otheroligonucleotide sequences.

All of the reactions involved were used above either in the preparationof d[ApC] 11 (B=adenin-9-yl, B′=cycsin-1-yl or of d[Gp(s)A] 13(E=adenin-9-yl. B′=adenin-9-yl) (Scheme 2).

First, the fully-protected dinucleoside phosphorothioate DMTr-Ap(s)C-Lev10 (B=14. B′=15) [ca. 0.75 mmol) was converted in four steps and in ca.96% overall isolated yield (Scheme 3a) into the partially-protectedtrimer 23. In Each coupling step, a ca. 20% excess of H-phosphonatemonomer 8 was used, but the excess of coupling agent 18 depended on thescale of the reaction. In addition a twofold excess of sulfur-transferagent 19 or 21 was used in this example. The products werechromatographed on silica gel after each “detritylation” step.

This material was then coupled with DMTr-Tp(H) 8 (B=17) and the productwas converted in three steps and in ca. 93% overall yield (Scheme 3b)into the fully-protected tetramer 24. The latter material was unblockedto give d[IpGp(s)ApC] 25 which was isolated without further purificationas its relatively pure (Ca. 96.5% by HPLC) sodium salt.

The tetranucleoside triphosphate d[TpGpApC] and the tetranucleosidetriphosphorothioate d[Cp(s)Tp(s)Gp(s)A] were also prepared by stepwisesynthesis in very much the same way. The protocols followed differedfrom that outlined in Scheme 3 only stepwise synthesis in very much thesame way. The protocols followed differed from that outlined in Scheme 3only inasmuch as the sulfur-transfer agent 19 was used exclusively inthe preparation of d[TpGpApC] and the sulfur-transfer agent 21 was usedexclusively in the preparation of d[Cp(s)Tp(s)Gp(s)A].

Reaction Scheme for Block Coupling

By way of illustration, Scheme 4 given herein-below illustrates anexample of block coupling which is part of the invention.

Reagents and Conditions

(i) N₂H₄H₂O, C₅H₅N-AcOH (3:1 v/v), 0° C., 20 min;

(ii) a, 30, Me₃C—COCl, C₅H₅N, −35° C., 30 min, b, Et₂N, H₂O;

(iii) 18 C₅H₅N, CH₂Cl₂, −35° C.;

(iv) a, 21, C₅H₅N, CH₂Cl₂, −35° C., 10 min, b, C₅H₅N—H₂O (1:1 v/v), −35°C. to room temp.

The fully protected octadeoxynucleoside heptaphosphorothioate 29 whichwas obtained in 91% isolated yield is a precursor ofd[Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T]. As indicated above, blockcoupling is much more feasible in solution than in solid phasesynthesis.

This approach is of course not in any way limited to tetramer coupling.Indeed, it is anticipated that this H-phosphonate approach will besuitable for coupling quite large oligonucleotide blocks (for example,10+10) together.

Reaction Scheme for Preparation of Block H-phosphonates

For example, partially-protected oligonucleotides 32a and thecorresponding phosphorothioates 33b which can be prepared by theconventional phosphotriester approved in solution (Chattopadhyaya, J.B.; Reese, C. B. Nucleic Acids Res., 1980, 8, 2039-2054; Kemal, O.,Reese. C. B.; Serafinowska, H. T. J. Chem. Soc., Chem. Commun., 1983,591-593) can similarly be converted into their 3′-H-phosphonates (34aand 34b, respectively) as indicated in Scheme 5.

Reagents and Conditions

(i) a, 30, Me₃C.COCl, C₅H₅N, −35° C., b, Et₃N, H₂O.

Example 1 Ac-Tp(s)Tp(s)Gp(s)G-OH

HO-Tp(s)Tp(s)Gp(s)G-Lev (5.82 g, 3 mmol) was co-evaporated withanhydrous pyridine (2×20 ml) and redissolved in anhydrous pyridine (30ml). Acetic anhydride (1.42 ml, 15 mmol) was added and the reactionsolution was allowed to stir at room temperature for 12 h. Water (1.5ml) was added to quench the reaction. After 10 min, the mixture wascooled to 0° C. (ice-water bath) and hydrazine hydrate (1.50 g, 30 mmol)in pyridine (15 ml) and glacial acetic acid (15 ml) was added. Themixture was stirred at 0° C. For 20 min and was then partitioned betweenwater (100 ml) and CH₂Cl₂ (100 ml). The two layers were separated andthe organic layer was washed with water (3×50 ml). The oroanic layer wasdried (MgSO₄) and evaporated. The residue was purified by silica gelchrorratooraphy. Impurities were eluted with methanol-dichloromethane(4:96 v/v) the main product was eluted with acetone. Evaporation of theappropriate fractions gave the partially protected tetradeoxynuciecsidetriphosphorothicate as colourless solid (5.30 g, 93%).

Example 2 Ac-Tp(s)Tp(s)Gp(s)Gp(H)

The ammonium salt of 4-methylphenyl H-phosphonate (1.42 g, 7.5 mmol) wasdissolved in the mixture of methanol (15 ml) and triethylamine (2.1 ml,15 mmol). The mixture vans evaporated and coevaporated with pyridine(2×10 ml) under reduced pressure. Ac-Tp(s)Tp(s)Gp(s)G-OH (4.71 g, 2.5mmol) was added and co-evaporated with dry pyridine (20 ml). The residuewas dissolved in dry pyridine (20 ml) and pivaloyl chloride (1.23 ml, 10mmol) was added at −35° C. in 1 min. After 30 min at the sametemperature, water (5 ml) was added and the mixture was allowed to warmto room temperature and stir for 1 hr. The solution was partitionedbetween water (100 ml) and dichloromethane (100 ml). The organic layerwas separated and washed with triethylammonium phosphate buffer (pH 7.0,0.5M, 3×50 ml), dried (MgSO₄), and then filtered and applied to a silicagel column (ca. 25 g). The appropriate fractions, which were eluted withmethanol-dichloromethane (20:80, v/v), were evaporated to giveAc-Tp(s)Tp(s)Gp(s)Gp(H), as a colourless solid (4.85 g, 94%).

Example 3 Ac-Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T-Bz

Ac-Tp(s)Tp(s)Gp(s)Gp(H) (1.229 g, 0.6 mmol) and HO-Gp(s)Gp(s)Tp(s)T-Bz(0.973 g, 0.5 mmol) were coevaporated with anhydrous pyridine (2×10 ml)and the residue was dissolved in anhydrous pyridine (10 ml). Thesolution was cooled to −35° C. (Industrial methylated spirits-dry icebath) and di-(2-chlorophenyl)phosphorochloridate (0.84 g, 2.5 mmol) indry dichloromethane (1 ml) was added over 10 min.4-[(2-Cyanoethyl)sulfanyl]morpholin-3,5-dione (0.20 g, 1.0 mmol) wasadded and the mixture was allowed to stir for 10 min at the sametemperature. Then water-pyridine (0.2 ml, 1:1 v/v) was added and themixture was stirred for a further 5 min. The reaction mixture was thenevaporated under reduced pressure. The residue was dissolved indichloromethane (100 ml) and the solution was washed with saturatedaqueous sodium bicarbonate solution (3×50 ml). The organic layer wasdried (MgSO4) and concentrated under reduced pressure. The residue waspurified by silica gel chromatography. Firstly, the lipophilicimpurities were removed with methanol-dichloromethane (4:96 v/v), andthen the main product was eluted with acetone. Evaporation of theappropriate fractions gave fully protected octadeoxynucleosideheptaphosphorothioate as colourless solid (1.81 g, 91%). Thefully-protected octadeoxynuclecside heptaphosphorothioate which wasobtained in 91% isolated yield is a precursor ofd[Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T].

1-13. (canceled)
 14. A process for introducing a group of formula —S-Ainto an H-phosphonate diester moiety, comprising: reacting a substratecomprising an H-phosphonate diester moiety with a sulfur transfer agentselected from the group consisting of a) compounds of formula:

wherein A represents an aryl, methyl, substituted alkyl or alkenylgroup; b) compounds of formula:

wherein A represents an aryl, methyl, substituted alkyl or alkenylgroup; and c) compounds of formula:

wherein A represents a methyl or alkenyl group.
 15. A process accordingto claim 14, wherein the sulfur transfer agent is selected from thegroup consisting of


16. A process according to claim 14 or claim 15, wherein theH-phosphonate diester is an oligonucleotide H-phosphonate diester.
 17. Aprocess according to claim 16, wherein the oligonucleotide H-phosphonatediester is a protected oligonucleotide H-phosphonate diester.
 18. Aprocess according to claim 17, further comprising one or more subsequentdeprotection steps, thereby producing a deprotected oligonucleotide. 19.A process for the preparation of an oligonucleotide, an oligonucleotidephosphorothioate or a mixed oligonucleotide/oligonucleotidephosphorothioate, said process comprising: introducing a group offormula —S-A into an H-phosphonate diester moiety by the processaccording to claim
 14. 20. A process according to claim 19, wherein thesulphur transfer agent is selected from the group consisting of


21. A compound of formula:

wherein A represents an aryl, methyl, substituted alkyl or alkenylgroup.
 22. A compound according to claim 21, wherein A represents aphenyl group or a —CH₂CH₂AN group.
 23. A compound according to claim 22,wherein A represents a phenyl group and the phenyl group isunsubstituted or is substituted by a halo or alkyl group.
 24. A compoundof formula:


25. A compound of formula:

wherein A represents an alkenyl group.